Nitric oxide releasing nanofiber stimulates revascularization in response to ischemia via cGMP-dependent protein kinase.

Nitric oxide (NO) promotes angiogenesis via various mechanisms; however, the effective transmission of NO in ischemic diseases is unclear. Herein, we tested whether NO-releasing nanofibers modulate therapeutic angiogenesis in an animal hindlimb ischemia model. Male wild-type C57BL/6 mice with surgically-induced hindlimb ischemia were treated with NO-releasing 3-methylaminopropyltrimethoxysilane (MAP3)-derived or control (i.e., non-NO-releasing) nanofibers, by applying them to the wound for 20 min, three times every two days. The amount of NO from the nanofiber into tissues was assessed by NO fluorometric assay. The activity of cGMP-dependent protein kinase (PKG) was determined by western blot analysis. Perfusion ratios were measured 2, 4, and 14 days after inducing ischemia using laser doppler imaging. On day 4, Immunohistochemistry (IHC) with F4/80 and gelatin zymography were performed. IHC with CD31 was performed on day 14. To determine the angiogenic potential of NO-releasing nanofibers, aorta-ring explants were treated with MAP3 or control fiber for 20 min, and the sprout lengths were examined after 6 days. As per either LDPI (Laser doppler perfusion image) ratio or CD31 capillary density measurement, angiogenesis in the ischemic hindlimb was improved in the MAP3 nanofiber group; further, the total nitrate/nitrite concentration in the adduct muscle increased. The number of macrophage infiltrations and matrix metalloproteinase-9 (MMP-9) activity decreased. Vasodilator-stimulated phosphoprotein (VASP), one of the major substrates for PKG, increased phosphorylation in the MAP3 group. MAP3 nanofiber or NO donor SNAP (s-nitroso-n-acetyl penicillamine)-treated aortic explants showed enhanced sprouting in an ex vivo aortic ring assay, which was partially abrogated by KT5823, a potent inhibitor of PKG. These findings suggest that the novel NO-releasing nanofiber, MAP3 activates PKG and promotes therapeutic angiogenesis in response to hindlimb ischemia.

Investigation of periodontitis, halitosis, xerostomia, and serological characteristics of patients with osteoarthritis and rheumatoid arthritis and identification of new biomarkers.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are two different types of arthritis. Within RA, the subsets between seronegative RA (snRA) and seropositive RA (spRA) represent distinct disease entities; however, identifying clear distinguishing markers between them remains a challenge. This study investigated and compared the oral health conditions in patients with RA and OA to clarify the differences from healthy controls. In addition, we investigated the serological characteristics of the patients, the factors that distinguished patients with RA from those with OA, and the main factors that differentiated between snRA and spRA patients. A total of 161 participants (mean age: 52.52 ± 14.57 years, 32 males and 129 females) were enrolled in this study and categorized as: normal (n = 33), OA (n = 31), and RA (n = 97). Patients with RA were divided into the following two subtypes: snRA (n = 18) and spRA (n = 79). Demographics, oral health, and serological characteristics of these patients were compared. The prevalence of periodontal diseases was significantly higher in patients with OA (100%) and RA (92.8%) than in healthy controls (0.0%). However, the presence of periodontal diseases was not utilized as a distinguishing factor between OA and RA. Xerostomia occurred more frequently in patients with RA (84.5%) than in patients with OA (3.2%) and healthy controls (0.0%) (all p < 0.001). ROC analysis revealed that periodontal disease was a very strong predictor in the diagnosis of OA compared to healthy controls, with an AUC value of 1.00 (p < 0.001). Additionally, halitosis (AUC = 0.746, 95% CI 0.621-0.871, p < 0.001) and female sex (AUC = 0.663, 95% CI 0.529-0.797, p < 0.05) were also significant predictors of OA. The strongest predictors of RA diagnosis compared to healthy controls were periodontal diseases (AUC = 0.964), followed by xerostomia (AUC = 0.923), age (AUC = 0.923), female sex (AUC = 0.660), and halitosis (AUC = 0.615) (all p < 0.05). Significant serological predictors of RA were anti-CCP Ab (AUC = 0.808), and RF (AUC = 0.746) (all p < 0.05). In multiple logistic regression analysis, xerostomia (odds ratio, OR: 8124.88, 95% CI 10.37-6368261.97, p-value = 0.008) and Anti-CCP Ab (OR: 671.33, 95% CI 2.18-207,074.02, p = 0.026) were significant predictors for RA compared to OA. When diagnosing spRA compared to snRA, anti-CCP Ab (AUC = 1.000, p < 0.001) and RF (AUC = 0.910, 95%CI 0.854-0.967, p < 0.001) had outstanding predictive performances. Therefore, clinicians and researchers should thoroughly evaluate the oral status of both OA and RA patients, alongside serological factors, and consider these elements as potential predictors.

Determination of pepsin in human saliva using pepsin-susceptible peptide reporter and colorimetric dipstick assay: a prospective, cross-sectional study.

A simple and effective pepsin detection assay is reported based on a pepsin-susceptible peptide (PSP) reporter degradation strategy. PSP, which can be specifically cleaved by pepsin, was modified with fluorescein isothiocyanate (FITC) and biotin at the N- and C-terminals to be used as a reporter for colorimetric detection of dipsticks. A universal lateral flow dipstick consisting of a streptavidin test line for biotin binding and a sample pad immobilized with a gold-labeled polyclonal (rabbit) anti-FITC antibody was used to verify PSP-based pepsin detection. When the PSP reporter reacts with pepsin in a tube, it cleaves into two fragments, and the cleaved fragments do not display any color on the test line. Therefore, the higher the concentration of pepsin is, the greater is the decrease in test line intensity (IT-line) and the higher is the control line intensity (IC-line). First, the PSP cleavage and dipstick assay conditions for pepsin detection was optimized. The ratio of color intensity (IT-line/IC-line) of PSP-based dipstick assay showed a linear relationship with log concentration of pepsin ranging between 4 and 500 ng/mL (R2 = 0.98, n = 6), with a limit of detection of 1.4 ng/mL. It also exhibited high specificity and good reproducibility. Finally, pepsin levels were quantified in saliva samples from healthy controls (n = 34) and patients with laryngopharyngeal reflux (LPR, n = 61). Salivary pepsin levels were higher in patients with LPR than in healthy controls. The salivary pepsin levels correlated with those measured using a conventional enzyme-linked immunosorbent assay kit. Therefore, this PSP-based dipstick assay is a convenient tool for assessing salivary pepsin levels.

Nanozyme based colorimetric detection of biogenic gaseous H2S using Ag@Au core/shell nanoplates with peroxidase-like activity.

A highly sensitive and facile colorimetric assay is introduced for detecting biogenic gaseous H2S using peroxidase (POD)-like catalytic activity of silver core/gold shell nanoplates (Ag@Au NPls). H2S can react with Ag@Au NPls to form Ag2S or Au2S on their surface, which can reduce POD-like activity of Ag@Au NPls and consequently decrease the absorbance at 650 nm due to oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) in the presence of hydrogen peroxide (H2O2). For in situ and multiple detection of H2S, we utilized a microplate cover with 24 polydimethylsiloxane inner wells where Ag@Au NPls reacted with H2S gas followed by treatment with TMB/H2O2. As a result, the change in absorbance at 650 nm showed a linear relationship with the H2S concentration in the range 0.33 to 2.96 µM (0.36 absorbance/µM H2S in PBS, R2 = 0.994) with a limit of detection of 263 nM and a relative standard deviation of 4.4%. Finally, this assay could detect H2S released from Eikenella corrodens, used as a model bacterium, in a short time (20 min) or at a low number of bacteria (1 × 104 colony forming units/mL). Therefore, this assay is expected to be applied for the study of H2S signaling in bacterial physiology, as well as measure H2S production released from other oral bacteria that cause halitosis and oral diseases, leading to the subsequent diagnosis.

Simple and Sensitive Detection of Bacterial Hydrogen Sulfide Production Using a Paper-Based Colorimetric Assay.

Hydrogen sulfide (H2S) is known to participate in bacteria-induced inflammatory response in periodontal diseases. Therefore, it is necessary to quantify H2S produced by oral bacteria for diagnosis and treatment of oral diseases including halitosis and periodontal disease. In this study, we introduce a paper-based colorimetric assay for detecting bacterial H2S utilizing silver/Nafion/polyvinylpyrrolidone membrane and a 96-well microplate. This H2S-sensing paper showed a good sensitivity (8.27 blue channel intensity/µM H2S, R2 = 0.9996), which was higher than that of lead acetate paper (6.05 blue channel intensity/µM H2S, R2 = 0.9959). We analyzed the difference in H2S concentration released from four kinds of oral bacteria (Eikenella corrodens, Streptococcus sobrinus, Streptococcus mutans, and Lactobacillus casei). Finally, the H2S level in Eikenella corrodens while varying the concentration of cysteine and treatment time was quantified. This paper-based colorimetric assay can be utilized as a simple and effective tool for in vitro screening of H2S-producing ability of many bacteria as well as salivary H2S analysis.

Electrochemical detection of uric acid in undiluted human saliva using uricase paper integrated electrodes.

In this study, we introduce a uricase-immobilized paper (UOx--paper) integrated electrochemical sensor for detection of uric acid (UA) in saliva. The UOx was immobilized on the detection zone in the wax-patterned paper substrate. This UOx-paper was integrated with a Prussian blue--modified, screen-printed carbon electrode after electropolymerization of o-phenylenediamine to construct an electrochemical cell for small-volume (20 µL) of samples. First, we optimized the fabrication conditions of UOx-paper. Next, the amperometric response of the UOx-paper-based electrochemical UA sensor was analyzed using a known concentration of UA standard solution in artificial saliva at an applied potential of - 0.1 V (versus Ag pseudo-reference electrode). The UOx--paper based electrochemical UA sensor showed a sensitivity of 4.9 µA·mM-1 in a linear range of 50 to 1000 µM (R2 = 0.998), high selectivity and good reproducibility, as well as a limit of detection of 18.7 µM (0.31 mg/dL) UA. Finally, we quantified the UA levels in undiluted saliva samples of healthy controls (n = 20) and gout patients (n = 8). The levels were correlated with those measured with conventional salivary UA enzymatic assays as well as serum UA levels. The UOx-paper-based electrochemical UA sensor is a user-friendly and convenient tool to assess salivary UA levels.

Adiponectin-targeted SERS immunoassay biosensing platform for early detection of gestational diabetes mellitus.

The anisotropic gold nanotriangles (AuNTs) were synthesized by a fast seedless growth process. The high-yield monodispersed AuNT colloids were obtained through a purification process based on depletion-induced interactions. AuNTs were modulated with a localized surface plasmon resonance (LSPR) peak of 638 nm wavelength coherent with the Raman excitation light. However, from finite element computation results, the AuNT clusters showed better performance for the 785 nm laser source due to a red shift in their LSPR properties, hence it was selected for the surface-enhanced Raman scattering (SERS) immunoassay. A self-assembly strategy using a thiol group and ON-OFF strategy in the heat map was performed to ensure the stability of SERS immunoassay platform. The sandwich SERS immunoassay biosensor platform for adiponectin detection demonstrated a wide assay range (10-15-10-6 g/mL), good reliability (R2 = 0.994, clinically relevant range), femto-scale limit of detection (3.0 × 10-16 g/mL), and excellent selectivity without interference from other biomarkers. This showed the possibility of effectively detecting adiponectin levels in the biofluids of pregnant women. Therefore, our technology is the first to quantitatively detect adiponectin based on SERS technology for early detection of gestational diabetes mellitus and has the potential to be used as a clinical biosensor capable of diagnosing various obstetric diseases during early pregnancy.

Cytotoxicity evaluation of sodium lauryl sulfate in a paper-based 3D cell culture system.

Because three-dimensional (3D) cell culture is more similar to in vivo cell microenvironments than two-dimensional (2D) cell culture, various 3D cell culture systems have been developed. Recently, paper has been used as a promising material for 3D cell culture and tissue models due to its flexibility, ease of manufacture, low cost, and widespread accessibility. In this study, we fabricated a paper-based 3D cell culture platform consisting of a hydrophilic region for cell attachment and a hydrophobic region printed with wax. Using this paper platform for 3D culture of L929 cells, we evaluated the cytotoxicity of a model substance, sodium lauryl sulfate (SLS), using water-soluble tetrazolium salt, Live/Dead, and luminescence assays. Then we compared those cytotoxicity results with results from a conventional 3D cell culture kit and 2D cell culture. We found that 3D cultured cells on paper responded more sensitively to SLS than 2D cultured cells, and the cytotoxicity of SLS to cells grown on the paper-based 3D cell culture platform was similar to that of cells grown using a commercially available 3D cell culture kit. Therefore, we expect that our paper-based 3D cell culture platform can be applied as a simple and facile tool for cell viability evaluation.

Analytical Methods for Detection of Gasotransmitter Hydrogen Sulfide Released from Live Cells.

Hydrogen sulfide (H2S) plays an important role in mammals as a signaling molecule. Recently, abnormal H2S concentration has been associated with several pathophysiological states, such as diabetes mellitus, hypertension, Alzheimer's disease, and Parkinson's disease. As regulating H2S concentration can be a very prominent way of developing new drugs, many researchers have paid great attention to H2S research. To understand the role of H2S in pathophysiology and develop H2S-based therapies, it is necessary to measure the exact concentration of H2S within biological systems. But, H2S is volatile and can be easily oxidized. Besides, the active sites for several biological effects of H2S are inside the cell. Therefore, there is a need for the development of new methods for the accurate and reliable detection of H2S within live cells. This review provides a summary of recent developments in H2S detection methods for live cell analysis.

Progress in Oral Microbiome Related to Oral and Systemic Diseases: An Update.

The human oral microbiome refers to an ecological community of symbiotic and pathogenic microorganisms found in the oral cavity. The oral cavity is an environment that provides various biological niches, such as the teeth, tongue, and oral mucosa. The oral cavity is the gateway between the external environment and the human body, maintaining oral homeostasis, protecting the mouth, and preventing disease. On the flip side, the oral microbiome also plays an important role in the triggering, development, and progression of oral and systemic diseases. In recent years, disease diagnosis through the analysis of the human oral microbiome has been realized with the recent development of innovative detection technology and is overwhelmingly promising compared to the previous era. It has been found that patients with oral and systemic diseases have variations in their oral microbiome compared to normal subjects. This narrative review provides insight into the pathophysiological role that the oral microbiome plays in influencing oral and systemic diseases and furthers the knowledge related to the oral microbiome produced over the past 30 years. A wide range of updates were provided with the latest knowledge of the oral microbiome to help researchers and clinicians in both academic and clinical aspects. The microbial community information can be utilized in non-invasive diagnosis and can help to develop a new paradigm in precision medicine, which will benefit human health in the era of post-metagenomics.

Rapid and simple colorimetric detection of hydrogen sulfide using an etching-resistant effect on silver nanoprisms.

A fast and sensitive colorimetric paper sensor has been developed using silver nanoprisms (Ag NPRs) with an edge length of ~50 nm for the detection of free H2S gas. We prepared two types of Ag NPRs-coated H2S sensing papers: a multi-zone patterned paper for passive (diffusion mode), and a single-zone patterned paper for pumped mode of H2S gas. The change in color intensity was quantitatively analyzed of Ag NPRs-coated paper after KCl treatment depending on the concentration of H2S gas, from yellow to purplish brown. As a result, Ag NPRs-coated H2S sensing paper showed good sensitivity with a linear range of 1.03 to 32.9 µM H2S, high selectivity, and good reproducibility and stability, together with a fast response time of 1 min. The developed H2S sensing paper was applied to detect the free H2S gas released from three types of garlic including crushed, peeled, and fresh garlic. Therefore, it can be utilized as a simple, fast, and reliable tool for on-site colorimetric detection of free H2S gas for quality control of dietary supplements and exhaled breath analysis.Graphical abstract.

Targeting hydrogen sulphide signaling in breast cancer.

INTRODUCTION: Hydrogen sulphide (H2S) has been established as a key member of the gasotransmitters family that recently showed a pivotal role in various pathological conditions including cancer. OBJECTIVES: This study investigated the role of H2S in breast cancer (BC) pathogenesis, on BC immune recognition capacity and the consequence of targeting H2S using non-coding RNAs. METHODS: Eighty BC patients have been recruited for the study. BC cell lines were cultured and transfected using validated oligonucleotide delivery system. Gene and protein expression analysis was performed using qRT-PCR, western blot and flow-cytometry. In-vitro analysis for BC hallmarks was performed using MTT, BrdU, Modified Boyden chamber, migration and colony forming assays. H2S and nitric oxide (NO) levels were measured spectrophotometrically. Primary natural killer cells (NK cells) and T cell isolation and chimeric antigen receptor transduction (CAR T cells) were performed using appropriate kits. NK and T cells cytotoxicity was measured. Finally, computational target prediction analysis and binding confirmation analyses were performed using different software and dual luciferase assay kit, respectively. RESULTS: The H2S synthesizing enzymes, cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CSE), exhibited elevated levels in the clinical samples that correlated with tumor proliferation index. Knock-down of CBS and CSE in the HER2+ BC and triple negative BC (TNBC) cells resulted in significant attenuation of BC malignancy. In addition to increased susceptibility of HER2+ BC and TNBC to the cytotoxic activity of HER2 targeting CAR T cells and NK cells, respectively. Transcriptomic and phosphoprotein analysis revealed that H2S signaling is mediated through Akt in MCF7, STAT3 in MDA-MB-231 and miR-155/ NOS2/NO signaling in both cell lines. Lastly, miR-4317 was found to function as an upstream regulator of CBS and CSE synergistically abrogates the malignancy of BC cells. CONCLUSION: These findings demonstrate the potential role of H2S signaling in BC pathogenesis and the potential of its targeting for disease mitigation.

Optimization of Saliva Collection and Immunochromatographic Detection of Salivary Pepsin for Point-of-Care Testing of Laryngopharyngeal Reflux.

Salivary pepsin is a promising marker for the non-invasive diagnosis of laryngopharyngeal reflux (LPR). For reliable results regarding pepsin in saliva, it is critical to standardize the collection, storage, and pre-processing methods. In this study, we optimized the saliva collection protocols, including storage conditions, i.e., solution, temperature, and time, and the pre-processing filter for pepsin. Moreover, we prepared a simple immunochromatographic strip for the rapid detection of pepsin and evaluated its sensing performance. As a result, we selected a polypropylene (PP) filter as the pre-processing filter for salivary pepsin in low resource settings, such as those where point of care testing (POCT) is conducted. This filter showed a similar efficiency to the centrifuge (standard method). Finally, we detected the pepsin using gold nanoparticles conjugated with monoclonal pepsin antibody. Under optimized conditions, the lower limit of detection for pepsin test strips was determined as 0.01 µg/mL. Furthermore, we successfully detected the salivary pepsin in real saliva samples of LPR patients, which were pre-processed by the PP filter. Therefore, we expect that our saliva collection protocol and pepsin immunochromatographic strip can be utilized as useful tools for a non-invasive diagnosis/screening of LPR in POCT.

Vaccine Efficacy Induced by 2009 Pandemic H1N1 Virus-Like Particles Differs from that Induced by Split Influenza Virus.

Influenza virus-like particles (VLPs) vaccines are highly immunogenic, showing strong protective efficacy against homologous virus infection compared to split vaccine. However, a comparative efficacy study against heterosubtypic virus infection between VLPs and split vaccine has yet to been reported. In this study, we generated VLPs vaccine containing hemagglutinin (HA) and matrix protein (M1) of the 2009 pandemic H1N1, and investigated the protective efficacies induced by VLPs vaccine and commercial monovalent H1N1 pandemic split vaccine from Sanofi-Pasteur. Mice were intramuscularly immunized with either VLPs vaccine or split vaccine and subsequently challenge-infected with homologous virus (A/California/04/2009, H1N1) or heterosubtypic virus (A/Philippines/82, H3N2) after 4.5 months. VLPs vaccination demonstrated a higher level of protective efficacy against homologous viruses compared to split vaccine, as lessened lung viral loads and minuscule levels of proinflammatory lung cytokines IFN-gamma and IL-6 were observed. Protective efficacies were close to non-existent in VLP-immunized mice challenged with heterosubtypic viruses (H3N2). In contrast, split vaccine showed lower vaccine efficacy against homologous virus than VLP vaccine, but conferred better protection against heterosubtypic viruses through lung viral loads reduction and heightened survival rate. These results indicate that influenza VLPs provide better protective efficacy against homologous virus challenge infection, whereas split vaccine shows better protective efficacy against heterosubtypic virus challenge. Findings from the current study contribute to the rational design of vaccines conferring a broad range of protection.

Hypoxia augments NaHS-induced ANP secretion via KATP channel, HIF-1α and PPAR-γ pathway.

It has been reported that sodium hydrosulfide (NaHS) stimulated high stretch induced-atrial natriuretic peptide (ANP) secretion via ATP sensitive potassium (KATP) channel. KATP channel is activated during hypoxic condition as a compensatory mechanism. However, whether NaHS affects ANP secretion during hypoxia remains obscure. The purpose of the present study is to discover the impact of NaHS on ANP secretion during hypoxia and to unravel its signaling pathway. Isolated beating rat atria were perfused with buffer exposed to different O2 tension (to 100% O2, normoxia; to 20% O2, hypoxia). The ANP secretion increased negatively correlated with O2 tension. NaHS (50 µM) did not show any significant effect on low stretch induced-ANP secretion in normoxic condition but augmented low stretch induced-ANP secretion in hypoxic condition. The augmentation of NaHS-induced ANP secretion during hypoxia was blocked by the pretreatment with KATP channel blocker (glibenclamide) and was enhanced by the pretreatment with KATP channel activator (pinacidil). Hypoxia increased the expression of PPAR-γ protein but did not change the expression of HIF-1α protein and eNOS phosphorylation. The NaHS-induced ANP secretion during hypoxia was also blocked by the pretreatment with HIF-1α inhibitor (2-methoxy- estradiol), PPAR-γ inhibitor (GW9662) but not by NOS inhibitor (L-NAME) and endothelin receptor inhibitor (bosentan). The intravenous infusion of NaHS increased plasma ANP level in monocrotaline-treated rats but not in sham rats. These results suggest that hypoxia augmented NaHS-induced ANP secretion partly through KATP channel, HIF-1α, and PPAR-γ pathway.

Development of an effective sample transfer device for biomarker detection in nasal secretions.

Nasal secretions (NS) reflect inflammatory activity of the nasal mucosa and thus can be utilized for disease diagnosis and determining treatment effects in Allergic rhinitis (AR). However, non-standardized collection of samples can affect the measured concentration of inflammatory biomarker in NS. In this study, we aimed to develop and evaluate new devices capable of standardizing the collection, storage, and preprocessing methods of NS samples. First, we chose the best swab as polyester (PE) and selected a stimulation method, twirling for 10 s at 1 Hz, to efficiently release AR biomarkers from a PE swab. Storage of sample solutions at -20 °C was optimal for the stability of biomarkers for the detection of AR. The new swab sample transfer device showed excellent concentration recovery efficiency (90-100%) for tryptase (Trp) and eosinophil cationic protein (ECP) without crosstalk between the two biomarkers. Finally, we compared the concentration of Trp in human NS samples of AR patients (n = 6) pre-processed by the new device with that by centrifuge as a standard method. As a result, the concentrations of Trp in NS were very similar in both groups. Therefore, this device can be utilized as an effective sample transfer and pre-processing device for point-of-care testing of AR.

An ultrasensitive electrochemical detection of tryptase using 3D macroporous reduced graphene oxide nanocomposites by one-pot electrochemical synthesis.

In this study, we introduce an ultrasensitive and stable sensing platform based on gold nanoparticle decorated 3D reduced graphene oxide - carbon nanotube nanocomposites (GNP/CNT/rGO) for detection of tryptase (Tryp), which is a potential biomarker within nasal secretions for allergic rhinitis (AR). The GNP/CNT/rGO on the glassy carbon electrode (GCE) was fabricated through single-step one-pot electrochemical co-reduction and deposition. The GNP/CNT/rGO exhibited excellent electrocatalytic activity as the working electrode compared with bare GCE, GNP/rGO and CNT/rGO on GCEs. The enhanced performance of GNP/CNT/rGO may be attributed to 3D macroporous structures, which may provide increased surface area, faster charge transfer and lower mass transport resistance, as well as high electrocatalytic activity and good biocompatibility due to GNP. A GNP/CNT/rGO-based sandwich-type electrochemical immunosensor was fabricated for detection of Tryp, which requires high sensitivity and specificity. The GNP/CNT/rGO-based immunosensor exhibited a detection range from 100 pg/mL to 100 ng/mL, a detection limit of 50 pg/mL and a sensitivity of 1.64 µA/(ng/mL). This immunosensor possessed high selectivity, excellent reproducibility (RSD 2.1%) and high stability over approximately 1 month. Therefore, we expect that the GNP/CNT/rGO and the modified immunosensor will be useful tools for detecting Tryp in nasal secretions for clinical diagnosis of AR.

Potential Protective Effect of Nitric Oxide-Releasing Nanofibers in Hypoxia/Reoxygenation-Induced Cardiomyocyte Injury.

Nitric oxide (NO) is involved in several physiological processes including vasodilation, angiogenesis, immune response, and wound healing, as well as preventing ischemia/reperfusion injury in many organs such as the heart, liver, lungs, and kidneys. Recently, various NO delivery systems such as nanoparticles, nanorods, and nanofibers have been widely studied as potential therapeutic agents. In particular, NO-releasing nanofibers have been attracting much attention for various medicinal applications including regenerative medicine, wound dressings, and coatings for implantable medical devices, due to their flexible and open architectures. In this study, we prepared biocompatible NO-releasing nanofibers by electrospinning using mixed solutions of polymers and methylaminopropyltrimethoxysilane (MAP3), which was modified with N-diazeniumdiolate as an NO donor. In addition, we evaluated their protective effects on hypoxia/reoxygenation (HR) injury in H9c2 cells. The total NO amount released from the resulting MAP3 nanofibers was 1.26 µmol ·mg-1. From the cytotoxicity evaluation of various weights of NO-releasing nanofibers (0 to 2 mg), we selected 1 mg NO-releasing nanofibers for the subsequent experiments. Pre-treatment with NO-releasing nanofibers before hypoxia induction could provide a cytoprotective effect against HR-induced injury in H9c2 cells. The nanofibers could also effectively inhibit the generation of hydrogen peroxide, which was one major contributor to oxidative damage, as well as 8-hydroxyl-2-deoxyguanosine level as an indicator of oxidative DNA damage. In addition, pre-treatment with NO-releasing nano-fibers in a wound model showed wound healing effects similar to those of normal cells. As a result, N-diazeniumdiolate-modified MAP3 nanofibers might protect H9c2 cells from DNA damage by inhibiting the generation of oxidative stress in HR injury. Therefore, we expect that NO-releasing nanofibers could be utilized as a therapeutic strategy for protecting cardiomyocytes from HR injury.

Characterization of H2S releasing properties of various H2S donors utilizing microplate cover-based colorimetric assay.

In this study, we characterized the potential H2S-releasing properties of seven different H2S donors, including sodium sulfide (Na2S), sodium hydrosulfide (NaHS), diallyl disulfide (DADS), diallyl trisulfide (DATS), sodium thiosulfate (Na2S2O3), morpholin-4-ium 4-methoxyphenyl-morpholino-phosphinodithioate (GYY4137), and Lawesson's reagent, in three assay solutions, phosphate buffered saline (PBS, pH 7.4), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffered saline (HBS, pH 7.6), and cell growth media (GM), utilizing our microplate cover-based colorimetric assay. For quantitative analyses of H2S-releasing characteristics of the various donors, we evaluated four parameters, maximum concentration of H2S at the steady state (Cmax), the time required to reach half of Cmax (t1/2), maximum releasing rate of H2S (Rmax), and time at H2S (tr-max). The results showed that the H2S-releasing kinetics of each H2S donor were dependent on the type of assay solution. In particular, the addition of GSH to DATS in GM released the fastest and highest amounts of H2S among the four H2S donors in the following order: DATS > DADS > Na2S ~ NaHS. The H2S-releasing characteristics of the H2S donors were well-matched with cell viability results of human prostate cancer PC-3 cells. Therefore, the microplate cover-based colorimetric assay will be a useful tool for accurate and efficient measurements of H2S-releasing dynamics.

Hydrogen sulfide donor, NaHS, stimulates ANP secretion via the KATP channel and the NOS/sGC pathway in rat atria.

Hydrogen sulfide (H2S) is normally produced from l-cysteine in mammalian tissues and related to the pathogenesis of cardiovascular diseases. The aim of this study is to investigate the effects of H2S donor on atrial natriuretic peptide (ANP) secretion and define its mechanism using normal and isoproterenol (ISP)-treated rats. Several H2S donors were perfused into isolated beating rat atria, and atrial pressure (AP) and ANP secretion were measured. NaHS augmented high stretch-induced ANP secretion and decreased AP in a dose-dependent manner. The high stretch-induced ANP secretion was stimulated by Na2S but was not changed by GYY4137 and sodium thiosulfate. NaHS and Na2S produced very high amount of H2S rapidly whereas GYY4137 produced very low amount of H2S slowly. NaHS-stimulated ANP secretion was blocked by the pretreatment with inhibitor for KATP channel, nitric oxide synthase (NOS), soluble guanylyl cyclase (sGC), phosphoinositol 3 kinase (PI3K) or protein kinase B. H2S synthesis enzyme inhibitor (DL-propargylglycine) did not show any significant changes in atrial parameters. However, the response of ANP secretion to NaHS markedly attenuated and DL-propargylglycine suppressed ANP secretion in ISP-treated rat atria. The expression of eNOS protein was decreased but the expression of cardiomyocyte-specific H2S producing enzyme, cystathione γ-lyase, was not changed in ISP-treated rat atria. The attenuation of NaHS-induced ANP secretion in ISP-treated rat atria may be due to the low expression of eNOS protein. These findings clarify that NaHS stimulates ANP secretion via the KATP channel and the PI3K/Akt/NOS/sGC pathway in rat atria.